The Role of TIMPs in Cell Growth and Differentiation: Tumor Angiogenesis

TIMP 在细胞生长和分化中的作用：肿瘤血管生成

基本信息

批准号：
8763694
负责人：
William Stetler-Stevenson
金额：
$ 62.9万
依托单位：
DIVISION OF CLINICAL SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8763694
关键词：
A549 ABCB1 gene ABCG2 gene AKR1C1 ATP-Binding Cassette Transporters Active Sites Acute Disease Alanine Amino Acids Angiogenesis Inhibition Angiogenesis Inhibitors Apoptosis Basement membrane Behavior Binding Biological Biological Assay Biological Response Modifier Therapy Breast Cancer Cell CD29 Antigen CD34 gene Cancer cell line Cell Differentiation process Cell Line Cell Proliferation Cell Surface Receptors Cell membrane Cell model Cell physiology Cell surface Cells Chemotherapy-Oncologic Procedure Chronic Disease Clinical Complex Cultured Tumor Cells Cyclic AMP Cyclic AMP-Dependent Protein Kinases Cyclic GMP Cytoskeleton Cytotoxic Chemotherapy Cytotoxic agent Data Development Diagnosis Disintegrins Dominant-Negative Mutation Doxorubicin Dyes E-Cadherin Elements Endothelial Cells Enzymes Epidermal Growth Factor Epithelial Equilibrium Extracellular Matrix Extracellular Matrix Degradation Extracellular Matrix Proteins Family Fibroblast Growth Factor Fibroblast Growth Factor 2 Fibroblasts Focal Adhesion Kinase 1 Future Gelatinase A Gene Expression Gene Expression Profiling Gene Expression Regulation Genes Glioblastoma Goals Growth Growth Factor Growth Inhibitors Human In Vitro Integrins Laboratories Ligands Lung Malignant Epithelial Cell Malignant neoplasm of lung Matrix Metalloproteinase Inhibitor Matrix Metalloproteinases Measures Mediating Mesenchymal Metalloproteases Methionine Methods Modeling NOD/SCID mouse Names Neoplasm Metastasis Nitric Oxide Donors Nitric Oxide Synthase Non-Small-Cell Lung Carcinoma Normal tissue morphology Pathology Pattern Phosphorylation Physiology Population Prognostic Factor Protein Dephosphorylation Protein Tyrosine Phosphatase Proteins Proto-Oncogene Proteins c-akt Publishing Radiation therapy Receptor Protein-Tyrosine Kinases Regulation Reporting Research Research Personnel Resistance Retroviral Vector Role S1-5 protein Series Side Signal Pathway Signal Transduction Solid Neoplasm Staging Stimulation of Cell Proliferation Survival Rate Testing Therapeutic Tissue Inhibitor of Metalloproteinase-1 Tissue Inhibitor of Metalloproteinases Tissues Topotecan Tumor Angiogenesis Tumor Cell Invasion Tumor Tissue Tyrosine Phosphorylation Vascular Endothelial Growth Factors Vascular Permeabilities Xenograft procedure Zinc angiogenesis arginyllysine base beta catenin cadherin 5 cancer cell cancer stem cell cancer therapy cell growth cell motility cell type density fibrosarcoma fibulin in vivo inhibitor/antagonist interest lung Carcinoma mRNA Expression member migration mutant neoplastic cell novel phospholipase C gamma prevent receptor research study response synthetic peptide tissue procollagenase tumor tumor growth tumor microenvironment tumor progression tumor xenograft tumorigenic vector control

项目摘要

During the past 4 years we have completed, (and published) several studies characterizing downstream signaling pathways from the TIMP-2-receptor, alpha3/beta1-integrin, involved in mediating the anti-angiogenic effects of TIMP-2, including a more detailed characterization of TIMP-2-mediated growth inhibition of endothelial cells (hMVECs) in response to fibroblast growth factor-2 (FGF-2) These studies demonstrated that inhibition of src homology protein tyrosine phosphatase-1 (SHP-1), either by pharmacologic methods or expression of dominant negative SHP-1, prevents TIMP-2-mediated inhibition of fibroblast growth factor mediated endothelial mitogenesis. These findings are consistent with previous observations that TIMP-2 activates SHP-1 resulting in inactivation (dephosphorylation) of a variety of receptor tyrosine kinases via a ligand-dependent fashion In another series of experiments, we examined the phosphorylation pattern of vascular endothelial growth factor-2 (VEGFR-2) following VEGF-A stimulation in the presence or absence of TIMP-2 (Ala+TIMP-2) Our results show that Ala+TIMP-2 selectively alters tyrosine phosphorylation of VEGFR-2 at residues implicated in endothelial cell proliferation and migration (significantly decreased phosphorylation at Y951, Y996 and Y1175). Ala+TIMP-2 disrupted downstream activation of phospholipase C-gamma, Akt and endothelial nitric oxide synthetase. TIMP-2 or Ala+TIMP-2 inhibit VEGF-A-mediated Ca+2 influx, and reduced cGMP levels normally enhanced by nitric oxide donors. The observed decrease in cGMP was sensitive to isobutylmethylxanthine inhibition. In another recent report we showed that TIMP-2 mediates the inhibition of vascular permeability via an alpha3/beta1-Shp-1-cAMP/PKA signaling pathway, which enhanced VE-cadherin association with the cytoskeleton Our results demonstrate the potential utility for TIMP-2 in cancer therapy through "normalization" of vascular permeability and function . It is this "normalization" of tumor vasculature, to which Jain and colleagues attribute much of the clinical benefit and enhanced response to radiotherapy following the use of angiogenesis inhibitors It is interesting that such "normalization" also seems to occur in vivo in our experiments utilizing forced expression of TIMP-2 in tumor xenografts (major unpublished observation, see below). We also completed our studies on the "B-C loop" synthetic peptides, identified by their alpha3/beta1-binding activity, demonstrating their anti-angiogenic and anti-tumorigenic activity in vivo. The proposed TIMP-2 mutants of the glutamic, lysine and arginine amino acid residues in the "B-C" loop region have been expressed but their activities have not yet been further characterized. As noted above these studies are incomplete because of staff departures and our focus on demonstrating in vivo MMP-independent activity of TIMP-2. As mentioned above our major focus has been to demonstrate the MMP-independent anti-angiogenic effects of TIMP-2 in contributing to the anti-tumor activity of TIMP-2 in vivo observed by a number of investigators. To this end we employed retroviral vectors to force expression of TIMP-2 and Ala+TIMP-2 in the human lung carcinoma cell line A549 and then used these cell lines in tumor xenograft experiments in both nu/nu and NOD-SCID mice. Although these cell lines showed no discernable difference in basal growth rates in vitro there was significant suppression of tumor growth in both TIMP-2 (90 %) and Ala+TIMP-2 (75%) xenografts compared to empty vector controls as late as 40 days post tumor-inoculation. The suppression of tumor growth was accompanied by a statistically significant decrease in tumor microvascular density count (CD 31+ or CD34+), a measure of antiangiogenic effects, as well as by increased tumor cell apoptosis (also possibly due to inhibition of angiogenesis). Somewhat unexpectedly, we also observed a decrease in focal adhesion kinase (FAK) in TIMP-2 expressing tumors and a significant decrease in FAK phosphorylation (Y397) in both TIMP-2 and Ala+TIMP-2 expressing tumor cells. Our observation that both FAK and/or AKT (Protein Kinase B, PKB) phosphorylation is reduced in TIMP-2 and Ala+TIMP-2 tumor tissues is significant in that: 1) FAK is upstream of AKT signaling, and both are involved in regulation of cell migration; 2) TIMP-2 and Ala+TIMP-2 expression reduced tumor cell migration in vitro. We previously reported decreased FAK phosphorylation in endothelial cells where it is involved in control of eNOS activity. In summary, these experiments using retrovirally transduced tumor cells expressing wild type (wt) TIMP-2 or metalloprotease inhibitor-deficient Ala+TIMP-2 clearly demonstrate that the MMP-independent activities of TIMP-2, including the anti-angiogenic activity, are of sufficient magnitude to significantly impact tumor growth in vivo. Our observation of the effects of TIMP-2 and Ala+TIMP-2 on A549 tumor xenografts, led us to perform transcriptional profiling of these cell lines and tumor tissues. The observed changes in gene expression are predominantly related to decreased tumor development and reduced metastasis In contrast to control A549 cells, cells expressing TIMP-2 or Ala+TIMP-2 showed increased expression of E-cadherin, and were resistant to redistribution of cell membrane associate E-cadherin and beta-catenin following epidermal growth factor (EGF) stimulation, suggestive of a mesenchymal-epithelial transition. Other genes of interest that were differentially regulated include EGF-containing fibulin-like extracellular matrix protein 1 (EGFEMP1, fibulin 3) that was up regulated in cells expressing TIMP-2 or Ala+TIMP-2. This protein is a favorable prognostic factor in gliomablastoma, and suppresses angiogenesis, cell proliferation and VEGF-A expression. However, these findings need to be confirmed (see below) and the mechanisms of the effects on downstream gene regulation remain to be identified. Additional data from our gene expression profiling also revealed changes in ATP-binding cassette (ABC) transporter gene expression. ABC proteins drive cell efflux of a variety of substrates, including cytotoxic drugs, and are known to contribute to resistance to cancer chemotherapy. The activity of ABC transporters is an important indicator of cancer stem cell (CSC) presence in various solid tumors. The Hoechst 33342 dye efflux assay identifies a tumor cell subpopulation, known as the side population (SP), that is enriched in CSCs. Based on our gene expression profiling data we posit that TIMP-2 anti-tumor activity may, in part, involve regulation of the SP in our lung cancer cell model. To this end, we determined the correlation between the SP fraction and level of endogenous TIMP-2 expression in a series of six non-small cell lung cancer (NSCLC) cell line. Interestingly, our results demonstrate a strong, highly significant inverse correlation (R2=0.073, p0.03) between the level of endogenous TIMP-2 mRNA expression and the percentage of SP determined using the Hoechst dye efflux assay. In A549 cells expressing TIMP-2, a significant decrease in the SP is observed and this decrease is associated with lower expression of ABCG2, ABCB1 and AKR1C1. Functional analysis reveals that A549 cells expressing TIMP-2 show increased sensitivity to cytotoxic drugs, including doxorubicin and topotecan. These findings suggest that TIMP-2 therapy may enhance sensitivity to cytotoxic chemotherapy, and are the first demonstration that TIMP-2 modulates SP and possibly CSC levels and function. We feel these studies demonstrate significant progress in developing a new biological activity that suppresses cancer stem cells, tumor cells, endothelial cells and tumor growth in vivo.

在过去 4 年中，我们完成（并发表）了几项研究，描述了 TIMP-2 受体、α3/β1-整合素下游信号通路的特征，涉及介导 TIMP-2 的抗血管生成作用，包括更详细的研究TIMP-2 介导的内皮细胞 (hMVEC) 生长抑制响应成纤维细胞生长因子 2 (FGF-2) 的特征这些研究表明，src 同源性的抑制蛋白酪氨酸磷酸酶-1 (SHP-1)，通过药理学方法或显性失活 SHP-1 的表达，可防止 TIMP-2 介导的对成纤维细胞生长因子介导的内皮有丝分裂的抑制。这些发现与之前的观察一致，即 TIMP-2 激活 SHP-1，通过配体依赖性方式导致多种受体酪氨酸激酶失活（去磷酸化）。在另一系列实验中，我们检查了血管内皮生长因子的磷酸化模式-2 (VEGFR-2) 在存在或不存在 TIMP-2 的情况下 VEGF-A 刺激后 (Ala+TIMP-2) 我们的结果表明，Ala+TIMP-2 选择性改变VEGFR-2 与内皮细胞增殖和迁移有关的残基上的酪氨酸磷酸化（Y951、Y996 和 Y1175 处的磷酸化显着降低）。 Ala+TIMP-2 破坏磷脂酶 C-gamma、Akt 和内皮一氧化氮合成酶的下游激活。 TIMP-2 或 Ala+TIMP-2 抑制 VEGF-A 介导的 Ca+2 内流，并降低通常由一氧化氮供体增强的 cGMP 水平。观察到的 cGMP 下降对异丁基甲基黄嘌呤抑制很敏感。在最近的另一份报告中，我们表明 TIMP-2 通过 alpha3/beta1-Shp-1-cAMP/PKA 信号通路介导血管通透性抑制，从而增强 VE-钙粘蛋白与细胞骨架的关联。我们的结果证明了 TIMP-2 的潜在效用2 通过血管通透性和功能的“正常化”进行癌症治疗。 Jain 及其同事将使用血管生成抑制剂后的大部分临床益处和对放疗的反应增强归因于肿瘤脉管系统的这种“正常化”。有趣的是，在我们利用血管生成抑制剂进行的实验中，这种“正常化”似乎也发生在体内。 TIMP-2 在肿瘤异种移植物中的强制表达（主要未发表的观察结果，见下文）。我们还完成了对“B-C 环”合成肽的研究，通过其 α3/β1 结合活性进行鉴定，证明了它们的体内抗血管生成和抗肿瘤生成活性。所提出的“B-C”环区中谷氨酸、赖氨酸和精氨酸氨基酸残基的TIMP-2突变体已被表达，但其活性尚未进一步表征。如上所述，由于工作人员离职以及我们专注于证明 TIMP-2 的体内 MMP 独立活性，这些研究并不完整。如上所述，我们的主要重点是证明 TIMP-2 的 MMP 独立抗血管生成作用有助于许多研究人员观察到的 TIMP-2 体内抗肿瘤活性。为此，我们采用逆转录病毒载体强制人肺癌细胞系 A549 中表达 TIMP-2 和 Ala+TIMP-2，然后在 nu/nu 和 NOD-SCID 小鼠的肿瘤异种移植实验中使用这些细胞系。尽管这些细胞系在体外基础生长率上没有明显差异，但与空载体对照相比，TIMP-2 (90%) 和 Ala+TIMP-2 (75%) 异种移植物中的肿瘤生长在晚至 40 年后仍显着受到抑制。肿瘤接种后几天。肿瘤生长的抑制伴随着肿瘤微血管密度计数（CD 31+ 或 CD34+）（抗血管生成作用的衡量标准）的统计显着下降，以及肿瘤细胞凋亡的增加（也可能是由于血管生成的抑制）。有点出乎意料的是，我们还观察到表达 TIMP-2 的肿瘤中粘着斑激酶 (FAK) 减少，并且表达 TIMP-2 和 Ala+TIMP-2 的肿瘤细胞中 FAK 磷酸化 (Y397) 显着减少。我们观察到 FAK 和/或 AKT（蛋白激酶 B，PKB）磷酸化在 TIMP-2 和 Ala+TIMP-2 肿瘤组织中均降低，其意义在于：1) FAK 是 AKT 信号传导的上游，并且两者都参与细胞迁移的调节； 2)TIMP-2和Ala+TIMP-2表达减少体外肿瘤细胞迁移。我们之前报道过内皮细胞中 FAK 磷酸化降低，FAK 参与 eNOS 活性的控制。总之，这些实验使用表达野生型（wt）TIMP-2 或金属蛋白酶抑制剂缺陷型 Ala+TIMP-2 的逆转录病毒转导肿瘤细胞，清楚地证明 TIMP-2 的 MMP 独立活性（包括抗血管生成活性）是足以显着影响体内肿瘤生长。我们观察了 TIMP-2 和 Ala+TIMP-2 对 A549 肿瘤异种移植物的影响，从而对这些细胞系和肿瘤组织进行了转录分析。观察到的基因表达变化主要与肿瘤发展减少和转移减少有关。与对照 A549 细胞相比，表达 TIMP-2 或 Ala+TIMP-2 的细胞显示 E-钙粘蛋白表达增加，并且对细胞膜重新分布具有抵抗力表皮生长因子 (EGF) 刺激后，E-钙粘蛋白和 β-连环蛋白相关，提示间质-上皮转变。其他受到差异调节的感兴趣基因包括含有 EGF 的 fibulin 样细胞外基质蛋白 1（EGFEMP1、fibulin 3），该蛋白在表达 TIMP-2 或 Ala+TIMP-2 的细胞中上调。该蛋白是胶质瘤母细胞瘤的有利预后因素，可抑制血管生成、细胞增殖和 VEGF-A 表达。然而，这些发现需要得到证实（见下文），并且对下游基因调控的影响机制仍有待确定。我们的基因表达谱的其他数据还揭示了 ATP 结合盒 (ABC) 转运蛋白基因表达的变化。 ABC 蛋白驱动多种底物（包括细胞毒性药物）的细胞流出，并且已知有助于抵抗癌症化疗。 ABC 转运蛋白的活性是各种实体瘤中癌症干细胞 (CSC) 存在的重要指标。 Hoechst 33342 染料流出检测可识别富含 CSC 的肿瘤细胞亚群，称为侧群 (SP)。根据我们的基因表达谱数据，我们推测 TIMP-2 抗肿瘤活性可能部分涉及肺癌细胞模型中 SP 的调节。为此，我们确定了一系列六种非小细胞肺癌 (NSCLC) 细胞系中 SP 分数与内源 TIMP-2 表达水平之间的相关性。有趣的是，我们的结果表明，内源性 TIMP-2 mRNA 表达水平与使用 Hoechst 染料流出测定法测定的 SP 百分比之间存在强烈且高度显着的负相关（R2=0.073，p0.03）。在表达 TIMP-2 的 A549 细胞中，观察到 SP 显着降低，这种降低与 ABCG2、ABCB1 和 AKR1C1 的表达降低有关。功能分析表明，表达 TIMP-2 的 A549 细胞对细胞毒性药物（包括阿霉素和拓扑替康）表现出更高的敏感性。这些发现表明 TIMP-2 疗法可能增强对细胞毒性化疗的敏感性，并且首次证明 TIMP-2 调节 SP 以及可能的 CSC 水平和功能。我们认为这些研究表明在开发抑制癌症干细胞、肿瘤细胞、内皮细胞和体内肿瘤生长的新生物活性方面取得了重大进展。